Complement is the collective term for a series of blood proteins that constitute a major effector mechanism of the immune system. The complement system plays an important role in the pathology of many autoimmune, inflammatory and ischemic diseases. Inappropriate complement activation and its deposition on host cells can lead to complement-mediated lysis and/or injury of cells and target tissues, as well as tissue destruction due to the generation of powerful mediators of inflammation. Key to the activity of the complement system is the covalent attachment of processed protein fragments derived from a serum protein, complement C3, to tissue sites of complement activation. This unusual property is due to the presence of a thioester bond in C3 that, when cleaved during C3 activation, converts C3 to a form designated C3b which can then utilize ester or amide bonds to link to cell and tissue-attached molecules. Once C3b is covalently attached, it is rapidly processed to the iC3b, C3dg and C3d forms, each of which remain covalently attached to the target tissue site. This process results in the “marking” of the tissue as one in which an inflammatory injury or other complement-related process is underway.
Complement can be activated by any of three pathways: the classical, lectin and alternative pathways. The classical pathway is activated through the binding of the complement system protein C1q to antigen-antibody complexes, pentraxins or apoptotic cells. The pentraxins include C-reactive protein and serum amyloid P component. The lectin pathway is initiated by binding of microbial carbohydrates to mannose-binding lectin or by the binding of ficolins to carbohydrates or acetylated molecules.
The alternative pathway is activated on surfaces of pathogens that have neutral or positive charge characteristics and do not express or contain complement inhibitors. This results from the process termed ‘tickover’ of C3 that occurs spontaneously, involving the interaction of conformationally altered C3 with factor B, and results in the fixation of active C3b on pathogens or other surfaces. The alternative pathway can also be initiated when certain antibodies block endogenous regulatory mechanisms, by IgA-containing immune complexes, or when expression of complement regulatory proteins is decreased. In addition, the alternative pathway is activated by a mechanism called the ‘amplification loop’ when C3b that is deposited onto targets via the classical or lectin pathway, or indeed through the tickover process itself, binds factor B. See Muller-Eberhard (1988) Ann. Rev. Biochem. 57:321. For example, Holers and colleagues have shown that the alternative pathway is amplified at sites of local injury when inflammatory cells are recruited following initial complement activation. Girardi et al., J. Clin. Invest. 2003, 112:1644. Dramatic complement amplification through the alternative pathway then occurs through a mechanism that involves either the additional generation of injured cells that fix complement, local synthesis of alternative pathway components, or more likely because infiltrating inflammatory cells that carry preformed C3 and properdin initiate and/or greatly increase activation specifically at that site.
Alternative pathway amplification is initiated when circulating factor B binds to activated C3b. This complex is then cleaved by circulating factor D to yield an enzymatically active C3 convertase complex, C3bBbn. C3bBb cleaves additional C3 generating C3b, which drives inflammation and also further amplifies the activation process, generating a positive feedback loop. Factor H is a key regulator (inhibitor) of the alternative complement pathway activation and initiation mechanisms that competes with factor B for binding to conformationally altered C3 in the tickover mechanism and to C3b in the amplification loop. Binding of C3b to Factor H also leads to degradation of C3b by factor I to the inactive form iC3b (also designated C3bi), thus exerting a further check on complement activation. Factor H regulates complement in the fluid phase, circulating at a plasma concentration of approximately 500 μg/ml, but its binding to cells is a regulated phenomenon enhanced by the presence of a negatively charged surface as well as fixed C3b, iC3b, C3dg or C3d. Jozsi et al., Histopathol. (2004) 19:251-258.
Complement activation, C3 fragment fixation and complement-mediated inflammation are involved in the etiology and progression of numerous diseases. The down-regulation of complement activation has been shown to be effective in treating several diseases in animal models and in ex vivo studies, including, for example, systemic lupus erythematosus and glomerulonephritis (Y. Wang et al., Proc. Nat'l Acad. Sci. USA (1996) 93:8563-8568), rheumatoid arthritis (Y. Wang et al., Proc. Nat'l Acad. Sci. USA (1995) 92:8955-8959), cardiopulmonary bypass and hemodialysis (C. S. Rinder, J. Clin. Invest. (1995) 96:1564-1572), hyperacute rejection in organ transplantation (T. J. Kroshus et al., Transplantation (1995) 60:1194-1202), myocardial infarction (J. W. Homeister et al., J. Immunol. (1993) 150:1055-1064; H. F. Weisman et al., Science (1990) 249:146-151), ischemia/reperfusion injury (E. A. Amsterdam et al., Am. J. Physiol. (1995) 268:H448-H457), antibody-mediated allograft rejection, for example, in the kidneys (J. B. Colvin, J. Am. Soc. Nephrol. (2007) 18(4):1046-56), and adult respiratory distress syndrome (R. Rabinovici et al., J. Immunol. (1992) 149:1744-1750).
Moreover, other inflammatory conditions and autoimmune/immune complex diseases are also closely associated with complement activation (B. P. Morgan. Eur. J. Clin. Invest. (1994) 24:219-228), including, but not limited to, thermal injury, severe asthma, anaphylactic shock, bowel inflammation, urticaria, angioedema, vasculitis, multiple sclerosis, myasthenia gravis, myocarditis, membranoproliferative glomerulonephritis, atypical hemolytic uremic syndrome, Sjögren's syndrome, renal and pulmonary ischemia/reperfusion, and other organ-specific inflammatory disorders. It is currently uncertain whether complement activation is essential to the pathogenesis and injury of all diseases in which local tissue C3 activation and inflammatory injury occurs; nevertheless, C3 fragment fixation is almost universally found as an associated event.
A variety of disorders are associated with inflammation, however, so definitive diagnosis of complement-mediated inflammation typically requires confirmation via immunostaining or other in vitro analysis performed on tissue samples retrieved by biopsy. While biopsies are in many respects routine, they have their limitations and are not risk-free. Because commonly used needle or punch biopsies sample only a small portion of the target organ, there is a risk of sample error leading to an incorrect diagnosis. Furthermore, although biopsy is a generally safe procedure, major complications such as internal bleeding may occur in a significant number of cases.
In some cases, because of the difficulties in diagnosing disease or monitoring disease progression, for example, in patients with systemic lupus erythematosus or lupus nephritis, repeat renal biopsies are therefore frequently necessary to assess the response to therapy or to diagnose disease relapse. See e.g., S. Bajaj et al., 2000, J. Rheumatol. 27:2822-2826. Although renal biopsy is generally a safe procedure, complications may occur in 6% or more of biopsies and intra-renal bleeding and hematuria are common Patients requiring repeat biopsies are at concomitantly greater risk of complications. See e.g., W. L. Whittier et al., 2004, J. Am. Soc. Nephrol. 15:142-147; D. C. Mendelssohn et al., 1995, Am. J. Kidney Dis. 26:580-585. Thus, a non-invasive method of detecting or accurately assessing the presence, degree and/or extent of complement-mediated inflammation would be of significant value in diagnosing disease, formulating treatment strategies and monitoring their efficacy for many inflammatory diseases, including lupus nephritis.
The use of complement receptor 2 (CR2), or functional fragments thereof, to target complement modulators to tissues which exhibit or express C3, or fragments of C3 to which the CR2 is able to bind, including C3b, iC3b, C3d and C3dg, is described in US 2008/0267980 and US 2008/0221011, the disclosures of which are hereby incorporated herein by reference. Such CR2 molecules, and functional fragments thereof, can be used for targeting because the first two N-terminal short consensus repeat domains (SCRs) comprise an active binding site for the exposed C3d domain that is contained within iC3b, C3dg, and C3d.
The present invention provides solutions to these and other problems in the art. The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.